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In this study, we investigate a gene augmentation therapy candidate for the treatment of retinitis pigmentosa (RP) due to cyclic nucleotide-gated channel beta 1 (CNGB1) mutations. We use an adeno-associated virus serotype 5 with transgene under control of a novel short human rhodopsin promoter. The promoter/capsid combination drives efficient expression of a reporter gene (AAV5-RHO-eGFP) exclusively in rod photoreceptors in primate, dog, and mouse following subretinal delivery. The therapeutic vector (AAV5-RHO-CNGB1) delivered to the subretinal space of CNGB1 mutant dogs restores rod-mediated retinal function (electroretinographic responses and vision) for at least 12 months post treatment. Immunohistochemistry shows human CNGB1 is expressed in rod photoreceptors in the treated regions as well as restoration of expression and trafficking of the endogenous alpha subunit of the rod CNG channel required for normal channel formation. The treatment reverses abnormal accumulation of the second messenger, cyclic guanosine monophosphate, which occurs in rod photoreceptors of CNGB1 mutant dogs, confirming formation of a functional CNG channel. In vivo imaging shows long-term preservation of retinal structure. In conclusion, this study establishes the long-term efficacy of subretinal delivery of AAV5-RHO-CNGB1 to rescue the disease phenotype in a canine model of CNGB1-RP, confirming its suitability for future clinical development., Competing Interests: Declaration of interests C.R.O. and A.F. are employees of Sanofi. S.M. is listed as inventor on the patent application WO2018172961A1 ‘‘Gene therapy for the treatment of cngb1-linked retinitis pigmentosa’’ and is co-founder of the gene therapy company ViGeneron GmbH., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The On-Off, or long flash, full field electroretinogram (ERG) separates retinal responses to flash onset and offset. Depending on degree of dark-adaptation and stimulus strength the On and Off ERG can be shaped by rod and cone photoreceptors and postreceptoral cells, including ON and OFF bipolar cells. Interspecies differences have been shown, with predominantly positive Off-response in humans and other primates and a negative Off-response in rodents and dogs. However, the rod signaling pathways that contribute to these differential responses have not been characterized. In this study, we designed a long flash protocol in the dog that varied in background luminance and stimulus strength allowing for some rod components to be present to better characterize how rod pathways vary from scotopic to mesopic conditions., Results: With low background light the rod a-wave remains while the b-wave is significantly reduced resulting in a predominantly negative waveform in mesopic conditions. Through modeling and subtraction of the rod-driven response, we show that rod bipolar cells saturate with dimmer backgrounds than rod photoreceptors, resulting in rod hyperpolarization contributing to a large underlying negativity with mesopic backgrounds., Conclusions: Reduction in rod bipolar cell responses in mesopic conditions prior to suppression of rod photoreceptor responses may reflect the changes in signaling pathway of rod-driven responses needed to extend the range of lighting conditions over which the retina functions., (© 2022. The Author(s).)

Purpose: Mutations in the cyclic nucleotide-gated (CNG) channel beta subunit (CNGB1) are an important cause of recessive retinitis pigmentosa. We identified a large animal model with a truncating mutation of CNGB1. This study reports the persistence of small, desensitized rod ERG responses in this model., Methods: Dark-, light-adapted and chromatic ERGs were recorded in CNGB1 mutant dogs and age and breed matched controls. Comparisons were made with a dog model known to completely lack rod function; young dogs with a mutation in the rod phosphodiesterase 6 alpha subunit (PDE6A -/- ). Immunohistochemistry (IHC) to label the rod CNG alpha (CNGA1) and CNGB1 subunits was performed., Results: The dark-adapted ERG of CNGB1 mutant dogs had a raised response threshold with lack of normal rod response and a remaining cone response. Increasing stimulus strength resulted in the appearance of a separate, slower positive waveform following the dark-adapted cone b-wave. With increasing stimulus strength this increased in amplitude and became faster to merge with the initial b-wave. Comparison of responses from PDE6A -/- (cone only dogs) with CNGB1 mutant dogs to red and blue flashes and between dark-adapted and light-adapted responses supported the hypothesis that the CNGB1 mutant dog had residual desensitized rod responses. CNGB1 mutant dogs had a small amount of CNGA1 detectable in the outer segments., Conclusions: CNGB1 mutant dogs have a residual ERG response from desensitized rods. This may be due to low levels of CNGA1 in outer segments., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objectives: To assess an inherited abnormal negative response electroretinogram (NRE) that originated in a family of Papillon dogs., Animals Studied: Thirty-eight dogs (Papillons, or Papillon cross Beagles or Beagles)., Procedures: Dogs underwent routine ophthalmic examination and a detailed dark-adapted, light-adapted and On-Off electroretinographic study. Vision was assessed using a four-choice exit device. Spectral-domain optical coherence tomography (SD-OCT) was performed on a subset of dogs. Two affected males were outcrossed to investigate the mode of inheritance of the phenotype., Results: The affected dogs had an increased underlying negative component to the ERG. This was most pronounced in the light-adapted ERG, resulting in a reduced b-wave and an exaggerated photopic negative response (PhNR). Changes were more pronounced with stronger flashes. Similarly, the On-response of the On-Off ERG had a reduced b-wave and a large post-b-wave negative component. The dark-adapted ERG had a significant increase in the scotopic threshold response (STR) and a significant reduction in the b:a-wave ratio. Significant changes could be detected at 2 months of age but became more pronounced with age. Vision testing using a four-choice device showed affected dogs had reduced visual performance under the brightest light condition. There was no evidence of a degenerative process in the affected dogs up to 8.5 years of age. Test breeding results suggested the NRE phenotype had an autosomal dominant mode of inheritance., Conclusions: We describe an inherited ERG phenotype in Papillon dogs characterized by an underlying negative component affecting both dark- and light-adapted ERG responses., (© 2022 The Authors. Veterinary Ophthalmology published by Wiley Periodicals LLC on behalf of American College of Veterinary Ophthalmologists.)

Pathogenic variants in retinol dehydrogenase 5 (RDH5) attenuate supply of 11-cis-retinal to photoreceptors leading to a range of clinical phenotypes including night blindness because of markedly slowed rod dark adaptation and in some patients, macular atrophy. Current animal models (such as Rdh5-/- mice) fail to recapitulate the functional or degenerative phenotype. Addressing this need for a relevant animal model we present a new domestic cat model with a loss-of-function missense mutation in RDH5 (c.542G > T; p.Gly181Val). As with patients, affected cats have a marked delay in recovery of dark adaptation. In addition, the cats develop a degeneration of the area centralis (equivalent to the human macula). This recapitulates the development of macular atrophy that is reported in a subset of patients with RDH5 mutations and is shown in this paper in seven patients with biallelic RDH5 mutations. There is notable variability in the age at onset of the area centralis changes in the cat, with most developing changes as juveniles but some not showing changes over the first few years of age. There is similar variability in development of macular atrophy in patients and while age is a risk factor, it is hypothesized that genetic modifying loci influence disease severity, and we suspect the same is true in the cat model. This novel cat model provides opportunities to improve molecular understanding of macular atrophy and test therapeutic interventions for RDH5-associated retinopathies., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Purpose: In this study, we assessed several extended electroretinographic protocols using nonstandard stimuli. Our aim was to separate and quantify the contributions of different populations of retinal cells to the overall response, both to assess normal function and characterize dogs with inherited retinal disease., Methods: We investigated three different protocols for measuring the full-field flash electroretinogram-(1) chromatic dark-adapted red and blue flashes, (2) increasing luminance blue-background, (3) flicker with fixed frequency and increasing luminance, and flicker with increasing frequency at a fixed luminance-to assess rod and cone contributions to electroretinograms recorded in phenotypically normal control dogs and dogs lacking rod function., Results: Temporal separation of the rod- and cone-driven responses is possible in the fully dark-adapted eye using dim red flashes. A- and b-wave amplitudes decrease at different rates with increasing background luminance in control dogs. Flicker responses elicited with extended flicker protocols are well fit with mathematical models in control dogs. Dogs lacking rod function demonstrated larger amplitude dark-adapted compared to light-adapted flicker responses., Conclusions: Using extended protocols of the full-field electroretinogram provides additional characterization of the health and function of different populations of cells in the normal retina and enables quantifiable comparison between phenotypically normal dogs and those with retinal disease., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: To analyze ERG responses from two dog models of retinitis pigmentosa, one due to a PDE6A mutation and the other a CNGB1 mutation, both to assess the effect of these mutations on retinal function and the ability of gene augmentation therapy to restore normal function., Methods: Scotopic and photopic ERGs from young affected and normal control dogs and affected dogs following AAV-mediated gene augmentation therapy were analyzed. Parameters reflecting rod and cone function were collected by modeling the descending slope of the a-wave to measure receptor response and sensitivity. Rod-driven responses were further assessed by Naka-Rushton fitting of the first limb of the scotopic b-wave luminance-response plot., Results: PDE6A -/- dogs showed a dramatic decrease in rod-driven responses with very reduced rod maximal responses and sensitivity. There was a minor reduction in the amplitude of maximal cone responses. In contrast, CNGB1 -/- dogs had some residual rod responses with reduced amplitude and sensitivity and normal cone responses. Following gene augmentation therapy, rod parameters were substantially improved in both models with restoration of sensitivity parameters log S and log K and a large increase in log R max in keeping with rescue of normal rod phototransduction in the treated retinal regions., Conclusions: Modeling of rod and cone a-waves and the luminance-response function of the scotopic b-wave characterized the loss of rod photoreceptor function in two dog models of retinitis pigmentosa and showed the effectiveness of gene augmentation therapy in restoring normal functional parameters., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Retinal diseases are common in dogs. Some hereditary retinal dystrophies in dogs are important not only because they lead to vision loss but also because they show strong similarities to the orthologous human conditions. Advances in in vivo non-invasive retinal imaging allow the capture of retinal cross-section images that parallel low power microscopic examination of histological sections. Spectral domain - optical coherence tomography (SD-OCT) allows the measurement of retinal layer thicknesses and gives the opportunity for repeat examination to investigate changes in thicknesses in health (such as changes with maturation and age) and disease (following the course of retinal degenerative conditions). The purpose of this study was to use SD-OCT to measure retinal layer thicknesses in the dog during retinal maturation and over the first year of life. SD-OCT was performed on normal beagle cross dogs from 4 weeks of age to 52 weeks of age. To assess changes in layer thickness with age, measurements were taken from fixed regions in each of the 4 quadrants and the area centralis (the region important for most detailed vision). Additionally, changes in retinal layer thickness along vertical and horizontal planes passing through the optic nerve head were assessed., Results: In the four quadrants an initial thinning of retinal layers occurred over the first 12 to 15 weeks of life after which there was little change in thickness. However, in the area centralis there was a thickening of the photoreceptor layer over this time period which was mostly due to a lengthening of the photoreceptor inner/outer segment layer. The retina thinned with greater distances from the optic nerve head in both vertical and horizontal planes with the dorsal retina being thicker than the ventral retina. Most of the change in thickness with distance from the optic nerve head was due to difference in thickness of the inner retinal layers. The outer retinal layers remained more constant in thickness, particularly in the horizontal plane and dorsal to the optic nerve head., Conclusions: These measurements will provide normative data for future studies.

Electroretinography (ERG) is a commonly used technique to study retinal function in both clinical and research ophthalmology. ERG responses can be divided into component waveforms, analysis of which can provide insight into the health and function of different types and populations of retinal cells. In dogs, ERG has been used in the characterization of normal retinal function, as well as the diagnosis of retinal diseases and measuring effects of treatment. While many components of the recorded waveform are similar across species, dogs have several notable features that should be differentiated from the responses in humans and other animals. Additionally, modifications of standard protocols, such as changing flash frequency and stimulus color, and mathematical models of ERG waveforms have been used in studies of human retinal function but have been infrequently applied to visual electrophysiology in dogs. This review provides an overview of the origins and applications of ERG in addition to potential avenues for further characterization of responses in the dog., (© 2020 American College of Veterinary Ophthalmologists.)